EP3562840A1 - Stabilisierter proteolytisch aktivierter wachstumsdifferenzierungsfaktor 11 - Google Patents

Stabilisierter proteolytisch aktivierter wachstumsdifferenzierungsfaktor 11

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Publication number
EP3562840A1
EP3562840A1 EP17832636.9A EP17832636A EP3562840A1 EP 3562840 A1 EP3562840 A1 EP 3562840A1 EP 17832636 A EP17832636 A EP 17832636A EP 3562840 A1 EP3562840 A1 EP 3562840A1
Authority
EP
European Patent Office
Prior art keywords
seq
polypeptide
protein
amino acids
gdfl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP17832636.9A
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English (en)
French (fr)
Inventor
R. Blake Pepinsky
Andreas Lehmann
Dingyi Wen
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Biogen MA Inc
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Biogen MA Inc
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Application filed by Biogen MA Inc filed Critical Biogen MA Inc
Publication of EP3562840A1 publication Critical patent/EP3562840A1/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Growth differentiation factor 11 is a member of the transforming growth- ⁇ (TGF- ⁇ ) family of proteins that play diverse roles in regulation of embryonic patterning and morphogenesis, cell proliferation and differentiation, adhesion, immune responses, cell growth arrest and tissue or organ regeneration and maintenance (Massague, Nature Rev. Mol. Cell Biol, 1 : 169-178 (2000); Chang et al, Endocr Rev. , 23:787-823 (2002); Heldin et al, Curr Opin Cell Biol , 21 : 166-76 (2009); Wakefield and Hill, Nat Rev Cancer, 13:328-41 (2013)).
  • TGF- ⁇ transforming growth- ⁇ family of proteins that play diverse roles in regulation of embryonic patterning and morphogenesis, cell proliferation and differentiation, adhesion, immune responses, cell growth arrest and tissue or organ regeneration and maintenance
  • This superfamily contains over 30 members, including TGFPs, activins, inhibins, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), glial cell line-derived neurotrophic factor family of ligands (GFLs), nodal and anti-Mullerian hormone (AMH) (Hinck, FEBS Lett. , 586: 1860- 70 (2012); Weiss and Attisano, Wiley Inter discip Rev Dev Biol, 2 ⁇ -63 (2013)). All of the proteins are expressed as large precursor proteins that undergo proteolytic processing to form non-covalently associated N-terminal pro- and C-terminal mature domains.
  • BMPs bone morphogenetic proteins
  • GDFs growth and differentiation factors
  • GFLs glial cell line-derived neurotrophic factor family of ligands
  • AH nodal and anti-Mullerian hormone
  • Processing of the precursors at mono and/or dibasic cleavage sites at the junction of the pro and mature domains can result in the formation of an active complex or a latent complex that requires further processing/activation steps.
  • Most members of the family form active complexes.
  • processing of GDFl l forms a latent complex.
  • GDFl l The mature domain of GDF11 is 109 amino acids in length and is not glycosylated. It consists of amino acids 299- 407, containing 9 cysteines that form 4 intramolecular disulfides and one interchain disulfide stabilizing the homodimer structure.
  • GDFl 1 contributes to early mesoderm development and anterior-posterior patterning of the axonal skeleton.
  • GDF11 has been identified to have roles in inhibiting neurogenesis in olfactory epithelium, preventing NGF-induced neurite outgrowth in PC 12 cells, and in promoting vascular remodeling and neurogenesis in animals treated with GDFl 1 (Ge et al, (supra); Katsimpardi et al, Science, 344:630-634 (2014)).
  • GDF11 it is highly desirable to obtain pharmaceutical formulations for use in vivo.
  • the disclosure features an isolated multimeric GDFl 1 protein comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide.
  • the isolated multimeric GDF11 protein comprises a first polypeptide, a second polypeptide, and a fourth polypeptide.
  • the multimeric GDFl 1 protein comprises a second polypeptide, a third polypeptide, and a fourth polypeptide.
  • the first polypeptide is non-covalently associated with the second polypeptide and the third polypeptide is non-covalently associated with the fourth polypeptide.
  • the second polypeptide is linked to the fourth polypeptide by a disulfide bond.
  • the first polypeptide and the third polypeptide each comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 60-1 12 of SEQ ID NO: 1
  • the second polypeptide and the fourth polypeptide each comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299-407 of human GDF11 (SEQ ID NO: l).
  • the first polypeptide and the third polypeptide can contain one to ten (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, insertions, and/or deletions within amino acids 60- 1 12 of SEQ ID NO: 1.
  • the second polypeptide and the fourth polypeptide can contain one to ten (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, insertions, and/or deletions within amino acids 296-407 or 299-407 of human GDFl 1 (SEQ ID NO: 1).
  • the substitutions are conservative amino acid substitutions.
  • the second polypeptide and the fourth polypeptide can contain one, two, or three deletions at the N- and/or C-terminus of amino acids 296-407 or 299-407 of human GDFl 1.
  • the multimeric protein can induce SMAD 2/3 phosphorylation in a Kinase Induced Receptor Activation Assay (KIRA).
  • the first polypeptide and the third polypeptide each comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 60-1 12 of SEQ ID NO: 1, and the second polypeptide and the fourth polypeptide each comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299-407 of human GDFl 1 (SEQ ID NO: 1).
  • the first polypeptide and the third polypeptide can contain one to ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, insertions, and/or deletions within amino acids 60-1 12 of SEQ ID NO: 1.
  • the second polypeptide and the fourth polypeptide can contain one to ten (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, insertions, and/or deletions within amino acids 296-407 or 299- 407 of human GDF1 1 (SEQ ID NO: 1).
  • the substitutions are conservative amino acid substitutions.
  • the second polypeptide and the fourth polypeptide can contain one, two, or three deletions at the N- and/or C-terminus of amino acids 299-407 of human GDF 11.
  • the multimeric GDF1 1 protein can induce SMAD 2/3 phosphorylation in a KIRA.
  • the first polypeptide and the third polypeptide are each at least 95% identical to amino acids 60- 112 of SEQ ID NO: 1.
  • the first polypeptide and the third polypeptide each consist of an amino acid sequence selected from the group consisting of amino acids 60- 112, 60-114, and 60-117 of SEQ ID NO: 1.
  • the first and third polypeptides consist of amino acids 60-112 and 60-1 14, respectively, or vice versa.
  • the first and third polypeptides consist of amino acids 60-1 12 and 60-1 17 of SEQ ID NO: 1, or vice versa.
  • the second polypeptide and the fourth polypeptide are each identical to amino acids 296-407 or 299-407 of SEQ ID NO: 1.
  • the first polypeptide and the third polypeptide each consist of amino acids 60- 112, 60-114, or 60-1 17 of SEQ ID NO: 1 and the second polypeptide and the fourth polypeptide each consist of amino acids 296-407 or 299-407 of SEQ ID NO: 1.
  • the asparagine at the position corresponding to position 94 of SEQ ID NO: 1 is glycosylated in each of the first polypeptide and the third polypeptide.
  • one or more of the polypeptides is linked to a half-life extending moiety.
  • the half-life extending moiety is PEG, XTEN, BSA, or an Fc moiety.
  • one or more of the polypeptides is linked to an agent that can traverse the blood brain barrier (e.g., FC5, FC5-Fc, anti-transferrin receptor antibody; insulin receptor antibody, insulin-like growth factor-1 receptor antibody, see e.g., Jones and Shusta, Blood Brain Transport of Therapeutics via Receptor Mediation, Pharm Res. 24: 1759-1771 (2007), incorporated by reference in its entirety herein).
  • the multimeric protein is in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • the pH of the pharmaceutical composition is in the range of 5.0 to 6.5. In other instances, the pH of the pharmaceutical composition is about 5.5. In certain instances, the protein remains soluble at pH 7.0.
  • the disclosure features an isolated protein comprising, in order, a first amino acid sequence and a second amino acid sequence linked directly via a linker (e.g., a peptide linker of 5 to 100 amino acids in length).
  • the first amino acid sequence is 52 to 65 amino acids in length and comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical, or 100% identical to amino acids 60 to 1 14 of SEQ ID NO: l, or amino acids 71-123 of SEQ ID NO: l .
  • the second amino acid sequence comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299-407 of SEQ ID NO: 1.
  • the first amino acid sequence can contain one to ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, insertions, and/or deletions within amino acids 60-1 14 of SEQ ID NO: 1.
  • the second amino acid sequence can contain one to ten (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, insertions, and/or deletions within amino acids 296-407 or 299- 407 of human GDF1 1 (SEQ ID NO: 1). In some instances, the substitutions are conservative amino acid substitutions.
  • the protein when activated by dimerization or by dimerization and proteolytic cleavage (e.g., with an endoproteinase) can induce SMAD 2/3 phosphorylation in a KIRA.
  • the first amino acid sequence is 52 to 62 amino acids in length (e.g., 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 amino acids in length). In other embodiments, the first amino acid sequence is 52 to 55 amino acids in length. In certain instances, the first amino acid sequence consists of amino acids 71-123, 60-122, or 60-1 14 of SEQ ID NO: 1. In some embodiments, the second amino acid sequence is at least 95% identical to amino acids 296-407 or 299-407 of SEQ ID NO: 1. In other embodiments, the second amino acid sequence consists of amino acids 296-407 or 299-407 of SEQ ID NO: l .
  • the peptide linker is 10 to 150, 10 to 144, 10 to 100, 10 to 50, 10 to 40, 10 to 30, 10 to 20, or 10 to 15 amino acids in length. In a specific embodiment, the peptide linker is 15 amino acids in length. In another specific embodiment, the peptide linker is 20 amino acids in length. In yet another specific embodiment, the peptide linker is 144 amino acids in length. In a specific embodiment, the peptide linker comprises or consists of G4S (SEQ ID NO: 4). In certain embodiment the serine in SEQ ID NO: 4 can be replaced with another amino acid. In another embodiment, the peptide linker lacks the GSG amino acid sequence.
  • the peptide linker is an XTEN (e.g., AE 144).
  • the protein comprises amino acids 35-211 of SEQ ID NO:6; amino acids 36- 217 of SEQ ID NO: 14; amino acids 36-227 of SEQ ID NO: 5; or amino acids 36-219 of SEQ ID NO:9.
  • the protein is linked to a half-life extending moiety. In certain instances, the half-life extending moiety is PEG, XTEN, HSA, or an Fc moiety.
  • the protein is linked to an agent that can traverse the blood brain barrier (e.g., FC5, FC5-Fc, anti-transferrin receptor antibody; insulin receptor antibody, insulin-like growth factor- 1 receptor antibody).
  • the protein is in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • the pH of the pharmaceutical composition is in the range of 5.0 to 6.5. In other instances, the pH of the pharmaceutical composition is about 5.5. In certain instances, the protein remains soluble at pH 7.0.
  • provided is a nucleic acid encoding the protein of this aspect.
  • expression vectors comprising the nucleic acid encoding the protein of this aspect are provided.
  • the disclosure encompasses isolated host cells comprising the nucleic acid or expression vector described above. Also provided are methods of making the protein of this aspect. The method involves culturing the host cell in a culture medium under conditions in which the protein is produced by the host cell (e.g., secreted into the culture medium). The method optionally involves isolating the protein. The isolated protein can be activated. In some embodiments, the protein is subjected to a disulfide reducing agent to create a first composition. The first composition is divided into a second and a third composition. The second composition is treated with or exposed to a cysteine activating agent to create a fourth composition. The fourth composition is combined with the third composition to create a fifth composition. In some instances, the fifth composition is active.
  • the fifth composition is treated with a protease that cleaves at the BMP1 site of the protein, thereby making an activated protein.
  • the disulfide reducing agent is DTT.
  • the cysteine activating agent is aldrithiol.
  • the protease that cleaves at the BMP1 site of the protein is endoproteinase AspN.
  • the method further involves formulating the fourth composition (if active) or fifth composition at a pH of 5.0 to 6.5. In a specific embodiment, the pH is 5.5.
  • the protein is produced by a mammalian cell (e.g., a CHO, COS, 293, NIH3T3 cell).
  • the protein is produced by a fungal cell (e.g., Aspergillus).
  • the protein is produced by an algal cell.
  • the disclosure provides an isolated multimeric GDF11 protein comprising a first polypeptide and a second polypeptide.
  • the first polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299- 407 of human GDF11 (SEQ ID NO: 1).
  • the second polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to any one of:
  • SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL (SEQ ID NO:21); SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPP (SEQ ID NO:28);
  • the first and/or second polypeptide can have one to ten amino acid substitutions, deletions, and/or insertions within amino acids 296-407 or 299-407 of SEQ ID NO: 1 or within SEQ ID NOs. : 21, or 28- 32.
  • the multimeric protein can induce SMAD 2/3 phosphorylation in a KIRA.
  • the first polypeptide consists of amino acids 296-407 or 299- 407 of SEQ ID NO: l.
  • the second polypeptide consists of the amino acid sequence of SEQ ID NO:21.
  • the first and/or second polypeptide is linked to a half-life extending moiety. In certain instances, the half-life extending moiety is PEG, XTEN, HSA, or an Fc moiety. In certain embodiments, the first and/or second polypeptide is linked to an agent that can traverse the blood brain barrier (e.g., FC5, FC5-Fc, anti-transferrin receptor antibody; insulin receptor antibody, insulin-like growth factor- 1 receptor antibody).
  • FC5, FC5-Fc anti-transferrin receptor antibody
  • insulin receptor antibody insulin-like growth factor- 1 receptor antibody
  • the protein is in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • the pH of the pharmaceutical composition is in the range of 5.0 to 6.5. In other instances, the pH of the pharmaceutical composition is about 5.5. In certain instances, the protein remains soluble at pH 7.0.
  • a pharmaceutical composition comprising a
  • the dimeric GDF1 1 proteins in the population comprise two GDF 11 monomers each of which consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 296- 407 or 299-407 of human GDF1 1 (SEQ ID NO: 1). At least 80% of the dimeric GDF l l proteins in the population comprise a polypeptide non-covalently associated with each GDF 11 monomer.
  • the polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 60-1 12 of SEQ ID NO: 1.
  • the GDF1 1 proteins can induce SMAD 2/3 phosphorylation in a KIRA.
  • At least 85% of the dimeric GDFl l proteins in the population comprise the polypeptide non-covalently associated with each GDF1 1 monomer. In some embodiments, at least 90% of the dimeric GDFl l proteins in the population comprise the polypeptide non-covalently associated with each GDFl l monomer. In some embodiments, at least 95% of the dimeric GDF1 1 proteins in the population comprise the polypeptide non- covalently associated with each GDFl l monomer. In some embodiments, at least 97% of the dimeric GDF1 1 proteins in the population comprise the polypeptide non-covalently associated with each GDFl l monomer.
  • the polypeptide non-covalently associated with each GDF l l mature domain monomer may be the same GDFl l propeptide polypeptide or different GDF l l propeptide polypeptides (e.g., 60-1 12 and 60-114; 60-112 and 60-1 17; or 60-1 14 and 60-117).
  • the polypeptide consists of amino acids 60- 112, 60-114, or 60-1 17 of SEQ ID NO: l and each GDFl l monomer consists of amino acids 296-407 or 299-407 of SEQ ID NO: l .
  • the asparagine at position 94 of SEQ ID NO: 1 is glycosylated in the polypeptide.
  • composition comprising a
  • the dimeric GDF 11 proteins in the population comprise two GDF 11 monomers each of which consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299- 407 of human GDF1 1 (SEQ ID NO: 1). At least 80% of the dimeric GDFl l proteins in the population comprise a polypeptide non-covalently associated with each GDFl 1 monomer.
  • the polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to: SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL (SEQ ID NO:21); SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPP (SEQ ID NO:28);
  • the dimeric GDFl 1 protein non- covalently associated with the polypeptide can induce SMAD 2/3 phosphorylation in a KIRA.
  • the polypeptide comprises or consists of the amino acid sequence:
  • SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL SEQ ID NO:21.
  • the asparagine at position 94 of SEQ ID NO: 1 is glycosylated in the polypeptide.
  • the disclosure provides a method of producing an activated human GDFl 1 protein.
  • the method involves contacting a GDFl 1 protein with a first protease that cleaves at a BMP1 site of the GDFl 1 protein; and then contacting the protein with a second protease (e.g., furin, plasmin, or trypsin).
  • a first protease that cleaves at a BMP1 site of the GDFl 1 protein
  • a second protease e.g., furin, plasmin, or trypsin
  • the GDFl l protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to a full length human GDFl 1 protein.
  • the GDFl 1 protein is a full length human GDFl 1 protein.
  • the human GDFl 1 protein is obtained from a mammalian cell (e.g., CHO, COS, 293, NIH3T3).
  • the human GDFl l protein is obtained from a yeast cell (e.g., Pichia pastoris). In certain embodiments, the human GDF11 protein is obtained from a microbial cell (e.g., E.coli). In certain embodiments, the human GDF11 protein is obtained from an algal cell. In certain embodiments, the human GDF11 protein is obtained from a fungal cell. In some
  • the protease that cleaves at the BMP1 site of the GDF11 protein is
  • the second protease is furin. In some embodiments, the second protease is plasmin. In some embodiments, the second protease is trypsin. In certain instances, the method further involves formulating the activated human GDFl l protein as a pharmaceutical composition.
  • the pharmaceutical composition is a sterile composition and has a pH in the range of 5.0 to 6.5. In other embodiments, the pharmaceutical composition is a sterile composition and has a pH of about 5.5.
  • this disclosure features a method of preparing a GDF11 protein formulation, the method comprising combining a first GDF11 polypeptide and a second GDF11 polypeptide.
  • the first GDF11 polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical, at least 98%, at least 99%, or 100% identical to:
  • SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL (SEQ ID NO:21); SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPP (SEQ ID NO:28);
  • the second GDFl l polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least
  • the first polypeptide comprises or consists of SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL (SEQ ID NO:21).
  • the second polypeptide consists of amino acids 299-407 of SEQ ID NO: 1.
  • the first GDF11 polypeptide is 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 amino acids in length.
  • the first and/or second polypeptide is/are linked to a half-life extending moiety (e.g., PEG, XTEN, HSA, Fc moiety).
  • the first and/or second polypeptide is linked to an agent that can traverse the blood brain barrier (e.g., FC5, FC5-Fc, anti-transferrin receptor antibody; insulin receptor antibody, insulin-like growth factor-1 receptor antibody).
  • the method further comprises adjusting the pH of the formulation to between 5.0 and 6.5. In a specific embodiment, the pH of the formulation is about 5.5. In certain embodiments, the formulation is a sterile formulation.
  • the disclosure features a method of treating a neurodegenerative disease in a human subject in need thereof.
  • the neurodegenerative disease is a disease of the central nervous system.
  • the disclosure features a method of treating a neurodegenerative disease in a human subject in need thereof.
  • the neurodegenerative disease is a disease of the central nervous system.
  • the disclosure features a method of treating a neurodegenerative disease in a human subject in need thereof.
  • the neurodegenerative disease is a disease of the central nervous system.
  • the central nervous system is a central nervous system.
  • neurodegenerative disease is a disease of the peripheral nervous system.
  • the method involves administering to the human subject a therapeutically effective amount of a protein(s) or a pharmaceutical composition described herein.
  • the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal Dementia, Lewy Body Dementia, Mild Cognitive Impairment, Posterior Cortical Atrophy, Primary Progressive Aphasia, Progressive Supranuclear Palsy, or Vascular Dementia.
  • the neurodegenerative disease is Alzheimer's disease.
  • the neurodegenerative disease is Parkinson's disease.
  • the neurodegenerative disease is Lewy Body Dementia.
  • the neurodegenerative disease is spinal muscular atrophy (SMA), myasthenia gravis, Isaacs syndrome, Stiff-Person syndrome, Guillian-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, amyotrophic lateral sclerosis, peripheral neuropathy, or thoracic outlet compression syndrome.
  • SMA spinal muscular atrophy
  • Isaacs syndrome Isaacs syndrome
  • Stiff-Person syndrome Stiff-Person syndrome
  • Guillian-Barre syndrome chronic inflammatory demyelinating polyneuropathy
  • amyotrophic lateral sclerosis amyotrophic lateral sclerosis
  • peripheral neuropathy or thoracic outlet compression syndrome.
  • the disclosure features a polypeptide comprising or consisting of the amino acid sequences set forth in any one of SEQ ID NO:21 or SEQ ID NO: 28-32.
  • these polypeptides have at least two (e.g., 2, 3, 4, 5, 6, 7, 8) amino acid substitutions.
  • the amino acids may be replaced by non-natural amino acids (e.g., non-natural amino acids that comprise olefinic side chains). These non-natural amino acids can form a staple(s) and/or stitch(es) under appropriate conditions, thereby forming stabilized or stapled peptides of these polypeptides.
  • the polypeptide is linked to a heterologous moiety (e.g., half-life extending moiety, a linker, and/or a moiety that can allow the polypeptide to traverse the blood brain barrier).
  • a heterologous moiety e.g., half-life extending moiety, a linker, and/or a moiety that can allow the polypeptide to traverse the blood brain barrier.
  • FIG.l is a schematic diagram denoting key features of the GDFl 1 sequence.
  • Signal peptide (residues 1-24), BMP1 type protease and furin-like convertase cleavage sites (arrows), N-terminal prodomain cleavage fragment (residues 25-122), C-terminal prodomain cleavage fragment (residues 123-298), furin recognition sequence (residues 295-298), mature domain (residues 299-407), polyalanine sequence (residues 29-41) and solubility enhancing sequence (residues 60-114, hatched pattern) within the N-terminal prodomain cleavage fragment, single N-linked glycosylation site (N94), single interchain disulfide forming cysteine (C372).
  • Figure discloses SEQ ID NO: 18.
  • FIG. 2A is a photograph of a SDS-PAGE gel used for characterization of GDF
  • GDFl 1 under reducing conditions.
  • the proteins were visualized by western blotting with a polyclonal antibody raised against the C-terminus of mature GDF11. Apparent molecular masses of molecular weight markers are indicated at the left. Arrows denote the stained bands of mature GDF1 1.
  • FIG. 2C is a photograph of a SDS-PAGE gel used for characterization of GDF11. Samples (5 ⁇ g/lane) were subjected to SDS-PAGE under reducing and non-reducing conditions on 4-20% gradient gels. The gel was stained with Coomassie brilliant blue.
  • Lane 1 full length GDF l 1 treated with plasmin for 2 hr at room temperature; lane 2, full length GDF l 1 treated with trypsin for 5 hr at room temperature; lane 3 full length GDFl 1 treated with furin for 24 hr at 37°C; lanes 4-7 full length GDF l 1 treated with endoproteinase Asp-N for 1.5 hr at 37°C; lane 4 AspN-treated GDFl 1 no further treatment; lane 5, AspN-treated GDF 11 treated with plasmin for 2 hr at room temperature; lane 6, AspN-treated GDF 11 treated with trypsin for 5 hr at room temperature; lane 7, AspN-treated GDF l 1 digested with furin for 24 hr at 37°C; lane 8, supernatant from lane 7sample; lane 9, pellet fraction from lane 7sample, lane 10 mature GDFl Istandard (1 ⁇ g); lane 1 1, supernatant from lane 4
  • FIG. 3A shows the characterization of GDFl 1 samples by size exclusion
  • Fractions 13-15 that correspond to lanes 3-5 were pooled and subject to analysis by mass spectrometry and activity measurements.
  • FIG. 3B shows the dissociation rate of the GDFl 1 prodomain peptide 60- 112/1 14/mature GDFl 1 complex as assessed by Octet.
  • the insert shows binding of the biotinylated GDFl 1 Asp-N/furin sample at 12.5 ⁇ g/mL, 25 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL, and 200 ⁇ g/mL.
  • FIG. 1 shows binding of the biotinylated GDFl 1 Asp-N/furin sample at 12.5 ⁇ g/mL, 25 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL, and 200 ⁇ g/m
  • FIG. 3C shows an Octet analysis of the binding characteristics of the GDFl 1 prodomain peptide 60-114/mature GDFl 1 complex, and AspN digested GDFl 1 for Activin RIB-Fc and Activin RIIA-Fc.
  • Biotinylated GDF11 samples (100 ⁇ g/mL) were captured on Streptavidin sensor tips for 5 min, washed for 1 min, then incubated for 15 min with the receptors (20 ⁇ g/mL), and dissociation over time was monitored for 5 min.
  • FIG. 4A is a graph showing the time course of GDFl 1 induced phosphorylation of
  • FIG. 4B is a graph depicting SMAD 2/3 phosphorylation activity of GDF11 test samples shown in Figure 2A following 60 min treatment. GDFl 1 was tested for function in a KIRA assay monitoring SMAD-2/3 phosphorylation.
  • FIG. 4C is a graph showing the signaling activity of GDF l 1 test samples shown in Figure 2 A in a luciferase reporter assay following 6 hr treatment.
  • GDFl 1 was tested for function in a SMAD reporter luciferase assay with luciferase expression under the control of the SMAD transcriptional response element (TRE).
  • FIG. 4D is a graph showing the signaling activity of GDFl 1 and TGF ⁇ 1 with and without treatment of GDF l 1 prodomain-Fc and TGF ⁇ 1 LAP-Fc in a luciferase reporter assay following 6 hr treatment.
  • GDFl 1 was tested for function in a SMAD reporter luciferase assay in which luciferase expression is under the control of the SMAD
  • FIG. 5A is a Reverse phase chromatography tracing of the reduced
  • FIG. 5B is a deconvoluted mass spectrum of the 25.0 min-peak from A.
  • FIG. 5C is a deconvoluted mass spectrum of the 21.3 min-peak from A.
  • FIG. 5D is a deconvoluted mass spectrum of the 22.1 min-peak from A.
  • FIG. 5E is a deconvoluted mass spectra of the reduced, deglycosylated, GDF l 1 treated with endoproteinase Asp-N shown in Figure 2A, lane 2.
  • FIG. 6A is a molecular model showing the association of the GDFl 1 solubility enhancing peptide on the crystal structure of mature GDFl 1.
  • the crystal structure of latent TGF- ⁇ (Shi et al, Nature, 474:343-9 (201 1)) was superposed on the GDFl 1 active mature domain (Padyana et al, Acta Crystallogr F Struc Biol Commun., 72: 160-164 (2016)) to position the GDFl 1 a2 helix by using the TGF- ⁇ a2 helix as an initial position.
  • the GDFl 1 al helix was docked against the GDFl 1 mature domain dimer using PIPER. See Example 1, Materials and Methods for more details on the model.
  • FIG. 6B is a molecular model of ACE490 propeptide-3xG4S (SEQ ID NO: 19;
  • FIG. 6C is a molecular model of ACE490 propeptide-3xG4S (SEQ ID NO: 19;
  • FIG. 6D is a schematic diagram showing AspN/furin cleavage-induced precipitation of prodomain peptide 122-299.
  • FIG. 7A shows a Coomassie blue stained SDS-PAGE analysis of conditioned medium for the fusion proteins indicated. Apparent molecular masses of molecular weight markers are indicated at the right. Arrow denotes the position of stained bands of prodomain/ mature GDFl 1 fusions. Samples were analyzed under non-reducing conditions.
  • FIG. 7B is a Western blot of fusions visualized using an anti-GDFl 1 C-terminal peptide polyclonal antibody for detection. Apparent molecular masses of molecular weight markers are indicated at the right. Samples were analyzed under reducing conditions.
  • FIG. 8A is a schematic diagram summarizing the redox steps used to induce dimerization of propeptide/mature GDFl 1 domain fusion proteins.
  • FIG. 8B is a Coomassie blue stained SDS-PAGE analysis of purified ACE490 proteins before and after redox under reducing and non-reducing conditions. Apparent molecular masses of molecular weight markers are indicated at the right. Lane 1, ACE490 alone; Lane 2, ACE490 plus DTT and AldrithiolTM; Lane 3, ACE490 plus DTT and
  • FIG. 8C is a graph showing signaling activity of ACE490 GDFl 1 test samples in luciferase reporter assay following 6 hr treatment.
  • FIG. 8D is a Coomassie blue stained SDS-PAGE analysis of purified ACE498 proteins before and after redox and after treatment with AspN under reducing and non- reducing conditions. Apparent molecular masses of molecular weight markers are indicated at the right. Lane 1, ACE498 alone; Lane 2, ACE498 plus DTT and AldrithiolTM for 20 hr at room temperature; Lane 3, ACE490 plus DTT/ AldrithiolTM and then treated with AspN; Lane 4, MW markers.
  • FIG. 8E is a graph depicting the signaling activity of ACE498 GDFl 1 test samples in luciferase reporter assay following 6 hr treatment.
  • FIG. 9A is a SDS-PAGE analysis of mature huGDFl 1 in the presence and absence of a synthetic propeptide.
  • FIG. 9B is a SDS-PAGE analysis of mature huGDFl 1 in the presence and absence of a synthetic propeptide.
  • FIG. 10A provides two secondary structure predictions for the indicated prodomain sequences of human GDFl 1 using PSIPRED (1999, Jones et al., Journal of Molecular Biology, 292, 195-202 (1999) and the more recent PSIPRED (2013, Buchan et al., Nucleic Acids Research, 41 (Wl): W340-W348 (2013).
  • " within the sequence indicates the first AspN cleavage site between LI 14 and Dl 15.
  • FIG. 11 shows synthetic peptides designed to form a complex with mature GDF11 ;
  • (*) put putative lasso helix, the putative helices (grey-shaded) follow the secondary structure predictions of PSIPRED (2013, Buchan et al, Nucleic Acids Research, 41
  • FIG. 12 provides secondary structure predictions for the first 132 residues of the human GFD11 pro-domain, according to PSIPRED (1999, Jones et al., Journal of Molecular Biology, 292, 195-202 (1999)) and PSIPRED (2013, Buchan et al, Nucleic Acids Research, 41 (Wl): W340-W348 (2013)) as in Figures 10A and 10B.
  • Figure discloses SEQ ID NO: 65.
  • Figure discloses SEQ ID NOS 66-68, respectively, in order of appearance.
  • FIG. 14 provides the results of an NCBI Protein Blast search for Protein Data Bank entries homologous to a putative helix sequence of human GDFl 1 prodomain peptide 92-107 (sequence APNISREVVKQLLPKA (SEQ ID NO:23)).
  • Figure discloses SEQ ID NOS 23, 25, 69-73, 70, 74-76, 33 and 77-89, respectively, in order of appearance.
  • FIG. 15 provides an alignment of GDF11 amino sequences from mouse, human, rat, zebra fish, chimpanzee, and cow. These alignments are helpful in determining which amino acids in a GDFl 1 sequence can be mutated without affecting the structure and/or activity of GDF 11. For example, amino acids at positions that are different in one or more of the GDF11 sequences can be substituted. The substitutions can be conservative substitutions.
  • proteolytic activation of full length GDFl 1 in vitro involves two steps: (i) initial cleavage at Asp- 122; and (ii) step (i) followed by cleavage at a cleavage site between the GDFl 1 prodomain and the GDFl 1 mature domain.
  • certain prodomain fragments of GDFl 1 can improve the solubility of the mature domain of GDFl 1 at neutral pH without inactivating the GDF 11 mature domain. Accordingly, this disclosure features solubility enhancing polypeptides of GDFl 1 that permit GDFl 1 polypeptides to be soluble at neutral pH and still remain active.
  • the GDFl 1 polypeptides are stored in an acid formulation, they can be administered to a human subject and the neutral pH blood would not render the GDF11 polypeptides insoluble.
  • the disclosure also provides methods of making such polypeptides and methods of using same.
  • GDF11 proteins from other species, e.g., cow, dog, cat, chicken, mouse, rat, pig, turkey, horse, fish, baboon, gorilla, are well known in the art (see, NCBI, Protein database). Methods for Activating GDFl 1 Proteins
  • the disclosure features a method for producing an activated GDFl 1 protein.
  • the method involves contacting a GDFl 1 protein with a first protease that cleaves at a BMP1 site of the GDFl 1 protein and a second protease that cleaves between the prodomain and the mature domain of GDFl 1.
  • the first protease and the second protease are added at the same or substantially the same time to the GDFl 1 protein that needs to be activated.
  • the first protease is added before adding the second protease to the GDFl 1 protein.
  • the GDFl 1 is human GDFl 1.
  • the human GDF l 1 has an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1.
  • the human GDFl 1 protein has one to ten (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, insertions, and/or deletions within SEQ ID NO: 1.
  • the GDF l 1 protein is linked to a heterologous moiety (e.g., a half-life extending moiety, a moiety that helps the protein traverse the blood brain barrier).
  • the first protease is an endoproteinase.
  • the protease that cleaves at the BMP1 site of the GDFl 1 protein is endoproteinase AspN.
  • the second protease is furin.
  • the second protease is plasmin.
  • the second protease is trypsin.
  • GDFl 1 When full length GDF l 1 proteins were recombinantly expressed in cells, the recombinant GDFl 1 protein was found to be inactive. In order to activate GDFl 1, the enzyme furin was added; however, furin was unable to promote cleavage at the cleavage site between the prodomain and the mature domain.
  • the GDFl 1 was first treated with a protease that can cleave at the aspartic acid residue at position of 122 of SEQ ID NO: 1, it was surprisingly found that GDFl 1 then became susceptible to cleavage at the cleavage site between the prodomain and the mature domain of GDFl 1.
  • an activated GDFl 1 protein wherein the GDFl 1 is expressed in a host cell.
  • the GDF l 1 from the host cell is isolated and contacted with a first protease that cleaves at a BMP1 site of the GDF 11 protein and a second protease that cleaves between the prodomain and the mature domain of GDF 11.
  • the first protease and the second protease are added at the same or substantially the same time to the GDFl 1 protein that needs to be activated.
  • the first protease is added before adding the second protease to the GDFl 1 protein.
  • the GDFl 1 is human GDFl 1.
  • the GDF 1 1 is human GDF 11.
  • the human GDF 11 has an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1.
  • the human GDF11 protein has one to ten amino acid substitutions, insertions, and/or deletions within SEQ ID NO: 1.
  • the first protease is an endoproteinase.
  • the protease that cleaves at the BMP 1 site of the GDF 11 protein is endoproteinase AspN.
  • the second protease is furin.
  • the second protease is plasmin. In yet other embodiments, the second protease is trypsin.
  • the host cell is a microbial cell (e.g., E. coli (see, e.g., Kamionka M, Curr Pharm Biotechnol, 12(2):268-274 (2001)). In certain instances, the host cell is a yeast cell (e.g., Pichia pastoris; Saccharomyces cerevisiae). In other embodiments, the host cell is an insect cell or a baculovirus-infected insect cell (see, e.g., Hu Y., Acta Pharmacol Sin.
  • the multimeric protein comprises two polypeptides comprising the mature domain of GDF 11 (e.g., 296-407 or 299-407 of SEQ ID NO: 1) and at least one (one or two) polypeptides comprising prodomain fragments of GDF11.
  • the prodomain fragments of GDF1 1 of the multimeric protein have the same amino acid sequence.
  • the prodomain fragments of GDF 11 of the multimeric protein are different (e.g., 60-1 12 and 60-1 14; 60-1 12 and 60-117; or 60-114 and 60-117 of SEQ ID NO: 1).
  • the multimeric GDF 11 protein comprises polypeptides from human GDF 11.
  • the mature domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the mature domain of human GDF11 - i.e., amino acids 296-407 or 299-407 of human GDFl 1 (SEQ ID NO: 1).
  • the mature domain comprises an amino acid sequence that is identical to the mature domain of human GDFl 1 (296-407 or 299-407 of SEQ ID NO: 1).
  • the polypeptide or polypeptides comprising prodomain fragments of GDFl 1 comprise or consist of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 60-112 of SEQ ID NO: 1.
  • the asparagine at the position corresponding to position 94 of SEQ ID NO: 1 is glycosylated in one or both polypeptides comprising prodomain fragments of GDF11.
  • the polypeptide or polypeptides comprising prodomain fragments of GDF11 comprise amino acids 60-112 of SEQ ID NO: l with at least one to ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, deletions, or insertions.
  • the polypeptide or polypeptides comprising prodomain fragments of GDFl 1 consist of amino acids 60-112 of SEQ ID NO: 1.
  • the polypeptide or polypeptides comprising prodomain fragments of GDF11 comprise amino acids 60-114 of SEQ ID NO: 1 with at least one to ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, deletions, or insertions.
  • the polypeptide or polypeptides comprising prodomain fragments of GDFl 1 consist of amino acids 60-114 of SEQ ID NO: l .
  • the polypeptide or polypeptides comprising prodomain fragments of GDFl 1 comprise amino acids 60-117 of SEQ ID NO: 1 with at least one to ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, deletions, or insertions.
  • the polypeptide or polypeptides comprising prodomain fragments of GDF11 consist of amino acids 60-117 of SEQ ID NO: 1.
  • the multimeric protein comprises prodomain fragments of GDF11 consisting of amino acids 60-112 and 60-117 of SEQ ID NO: l .
  • the multimeric protein comprises prodomain fragments of GDFl 1 consisting of amino acids 6-112 and 60-114 of SEQ ID NO: 1. In certain cases, the multimeric protein comprises prodomain fragments of GDF11 consisting of amino acids 6- 114 and 60-117 of SEQ ID NO: 1.
  • the first polypeptide comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to amino acids 296-407 or 299-407 of human GDF11 (SEQ ID NO: 1).
  • the first polypeptide has one to ten (one, two, three, four, five, six, seven, eight, nine, or ten) substitutions, deletions or insertions in amino acids 296-407 or 299-407 of human GDF11 (SEQ ID NO: 1).
  • the cysteine residues in the mature domain are not altered.
  • one to three amino acids are deleted at the N and/or C-terminus of amino acids 296-407 or 299-407 of human GDF11 (SEQ ID NO: 1).
  • one to five amino acids are substituted within amino acids 296-407 or 299-407 of human GDF11 (SEQ ID NO: 1).
  • the substitutions may be conservative or non-conservat ve.
  • the first polypeptide can be 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90 amino acids in length.
  • the second polypeptide comprises RELRLESIKSQILSKLRLKG (SEQ ID NO:51) or a stabilized peptide thereof.
  • the second polypeptide can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
  • the second polypeptide comprises or consists of SPRELRLESIKSQILSKLRLKG (SEQ ID NO:32) or a stabilized peptide thereof.
  • the second polypeptide comprises or consists of SPRELRLESIKSQILSKLRLKEAPNIS (SEQ ID NO:29) or a stabilized peptide thereof.
  • the second polypeptide comprises or consists of
  • the second polypeptide comprises or consists of
  • the second polypeptide comprises or consists of SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPP (SEQ ID NO:28) or a stabilized peptide thereof.
  • the second polypeptide comprises or consists of SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL (SEQ ID NO:21) or a stabilized peptide thereof.
  • the stabilized peptide can be made by replacing at least two (e.g., 2, 3, 4, 5, 6, 7, or 8) amino acids of the above-listed sequences that are separated by 2, 3, or 6 amino acids with non-natural amino acids with olefin-containing side chains that can be covalently joined, e.g., using a ring-closing metathesis reaction.
  • two amino acids (separated by 2, 3, or 6 amino acids) of each of the above sequences is replaced with non-natural amino acids with olefin-containing side chains.
  • One or more of the polypeptides of the multimeric protein described above can be linked to a half-life extending moiety. Such half-life extending moieties are discussed further below.
  • polypeptides of the multimeric protein described above can be linked to a moiety that can assist the multimeric protein traverse the blood brain barrier. Such moieties are discussed further below.
  • the activity of the multimeric GDF11 protein can be assayed according to any method known in the art. In one instance, the activity of the multimeric GDF11 protein is assayed using the Kinase Induced Receptor Activation Assay. In another embodiment, the activity of the multimeric GDF 11 protein is assayed using reporter assays in SMAD2/3 reporter cells. In yet another embodiment, the activity of the multimeric GDF1 1 protein is assayed using a SMAD2/3 phosphorylation assay.
  • fusion proteins linking a prodomain fragment of GDF1 1 with the mature domain of GDF 11.
  • the two sequences of GDF 11 can be linked or conjugated together by any method known in the art, including the use of peptide linkers.
  • the fusion protein can comprise, in order, a first GDF1 1 amino acid sequence and a second GDF1 1 amino acid sequence linked directly via a peptide linker of 5 to 150 amino acids in length.
  • the peptide linker is 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 100, 10 to 144, or 10 to 150 amino acids in length.
  • the linker is 15 amino acids in length.
  • the linker is 20 amino acids in length.
  • the linker is 144 amino acids in length. Linkers are discussed further below.
  • the first amino acid sequence is 52 to 65 amino acids in length and comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 60 to 1 14 of SEQ ID NO: 1.
  • the first amino acid sequence is 52 to 65 amino acids in length and comprises amino acids 60 to 114 of SEQ ID NO: 1 with one to ten amino acid substitutions, deletions, and/or insertions. In certain instances, one to six amino acids of the first amino acid sequence are substituted with non-natural amino acids.
  • the first amino acid sequence is 52 to 65 amino acids in length and comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 71 to 123 of SEQ ID NO: l .
  • amino acids 71 to 123 of SEQ ID NO: l are examples of amino acids 71 to 123 of SEQ ID NO: l .
  • the first amino acid sequence is 52 to 65 amino acids in length and comprises amino acids 71 to 123 of SEQ ID NO: 1 with one to ten amino acid substitutions, deletions, and/or insertions. In some embodiments, the first amino acid sequence is 52 to 62 amino acids in length. In some embodiments, the first amino acid sequence is 52 to 55 amino acids in length. In some embodiments, the first amino acid sequence is 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acids in length. In one embodiment, the first amino acid sequence consists of amino acids 71-123 of SEQ ID NO: l .
  • the first amino acid sequence consists of amino acids 60-122 of SEQ ID NO: 1. In yet another embodiment, the first amino acid sequence consists of amino acids 60-114 of SEQ ID NO: 1. In certain embodiments, the first amino acid sequence is a stabilized polypeptide (e.g., a stapled polypeptide based on amino acids 60-114 of SEQ ID NO: 1, or amino acids 71-123 of SEQ ID NO: 1, or amino acids 60-122 of SEQ ID NO: 1). In certain embodiments, the first amino acid sequence comprises an alpha-helical region from a non-GDFl l protein. In such instances, the first amino acid sequence may be 52 to 65 amino acids in length.
  • a stabilized polypeptide e.g., a stapled polypeptide based on amino acids 60-114 of SEQ ID NO: 1, or amino acids 71-123 of SEQ ID NO: 1, or amino acids 60-122 of SEQ ID NO: 1).
  • the first amino acid sequence comprises an alpha-helical region from a non-G
  • the second amino acid sequence comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299-407 of SEQ ID NO: l.
  • the second amino acid sequence comprises or consists of amino acids 296-407 or 299-407 of SEQ ID NO: 1 with one to ten amino acid substitutions, insertions, or deletions.
  • the fusion protein comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 35-211 of SEQ ID NO:6.
  • the fusion protein comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to 36-219 of SEQ ID NO:9.
  • the fusion proteins described herein may require activation by dimerization, or by both dimerization and proteolytic cleavage.
  • the fusion protein is subjected to a disulfide reducing agent to create a first composition.
  • the first composition is then divided into a second and a third composition.
  • the second composition is contacted with a cysteine activating agent to create a fourth composition.
  • the fourth composition is then combined with the third composition to create a fifth composition.
  • the fifth composition can be tested for activity as described above. If the fifth composition is not active or has low activity, the fifth composition can be treated with a protease that cleaves at the BMP1 site of the protein, thereby making an activated protein. In certain embodiments, the fifth composition is active.
  • the disulfide reducing agent is dithiothreitol (DTT).
  • the cysteine activating agent is aldrithiol.
  • the protease that cleaves at the BMP1 site of the protein is endoproteinase AspN.
  • the composition comprising the activated protein may be formulated as a sterile pharmaceutical composition. In certain instances, the formulation has a pH of about 5.0 to about 5.5. In certain instances, the formulation has a pH of about 5.5.
  • the fusion proteins described herein are produced recombinantly in host cells.
  • the host cell is a microbial cell (e.g., E. coli).
  • the host cell is a yeast cell (e.g., Pichia pastoris; Saccharomyces cerevisiae).
  • the host cell is an insect cell or a baculovirus-infected insect cell.
  • the host cell is a mammalian cell (e.g., CHO, COS, 293, NIH 3T3 cells).
  • the host cell is a filamentous fungal cell (e.g., Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei).
  • the host cell is an algal cell, such as a microalgal cell (e.g., Chlamydomonas reinhardtii, Scenedesmus obliquus).
  • the fusion proteins described above can be linked to a half-life extending moiety. Such half-life extending moieties are discussed further below.
  • the fusion proteins described above can also be linked to a moiety that can assist the protein traverse the blood brain barrier. Such moieties are discussed further below.
  • the multimeric protein or chimeric proteins described herein can be formulated as a pharmaceutical composition.
  • the pharmaceutical composition is a sterile formulation that has a pH of about 5.0 to about 5.5.
  • the pharmaceutical composition is a sterile formulation that has a pH of about 5.5.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a population of dimeric GDF l 1 proteins.
  • the dimeric GDFl 1 proteins in the population comprise two GDF11 monomers each of which consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299-407 of human GDF11 (SEQ ID NO: 1).
  • the GDF l 1 monomers have one to ten substitutions, insertions, and/or deletions in amino acids 296-407 or 299-407 of human GDFl 1 (SEQ ID NO: 1). In some cases, none of the cysteine residues of each monomer is substituted or deleted. In some case one to three amino acids are deleted at the N- and/or C-terminus of amino acids 299-407 of human GDFl 1 (SEQ ID NO: 1). At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% of the dimeric GDFl 1 proteins in the population comprise a polypeptide non-covalently associated with each GDF 11 monomer.
  • the polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 60-1 12 of SEQ ID NO: 1.
  • compositions are active.
  • they can induce SMAD 2/3
  • the polypeptide consists of amino acids 60-112 of SEQ ID NO: 1 and each GDF1 1 monomer consists of amino acids 296-407 or 299-407 of SEQ ID NO: 1.
  • the polypeptide consists of amino acids 60-114 of SEQ ID NO: 1 and each GDF1 1 monomer consists of amino acids 296-407 or 299-407 of SEQ ID NO: 1.
  • the polypeptide consists of amino acids 60-117 of SEQ ID NO: l and each GDF1 1 monomer consists of amino acids 296-407 or 299-407 of SEQ ID NO: 1.
  • the asparagine at position 94 of SEQ ID NO: 1 is glycosylated in the polypeptide.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a population of dimeric GDF 11 proteins.
  • the dimeric GDF1 1 proteins in the population comprise two GDF11 monomers each of which consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299-407 of human GDF11 (SEQ ID NO: 1).
  • the GDF 11 monomers have one to ten substitutions, insertions, and/or deletions in amino acids 296-407 or 299-407 of human GDF1 1 (SEQ ID NO: 1). In some cases, none of the cysteine residues of each monomer is substituted or deleted. In some case one to three amino acids are deleted at the N- and/or C-terminus of amino acids 296-407 or 299-407 of human GDF1 1 (SEQ ID NO: 1). At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% of the dimeric GDF 11 proteins in the population comprise a polypeptide non-covalently associated with each GDF 11 monomer.
  • the polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO:21 , SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.
  • the population of GDF1 1 proteins of the pharmaceutical composition are active. For example, they can induce SMAD 2/3 phosphorylation in a Kinase Induced Receptor Activation Assay.
  • the asparagine at position 94 of SEQ ID NO: 1 is glycosylated in the polypeptide.
  • the disclosure also provides a method of preparing a GDF 11 protein formulation comprising a first polypeptide and a second polypeptide to improve solubility.
  • the first polypeptide can be a polypeptide that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO:21 , SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.
  • the first polypeptide is 22 to 50 amino acids in length.
  • the first polypeptide is a stapled peptide (e.g., a hydrocarbon-stapled peptide). In certain cases, the first polypeptide is a non- GDF 11 polypeptide that comprises an alpha-helical sequence.
  • the second polypeptide comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 296-407 or 299-407 of human GDF1 1 (SEQ ID NO: 1).
  • the second polypeptide has one to ten substitutions, insertions, and/or deletions in amino acids 296-407 or 299-407 of human GDF 11 (SEQ ID NO: 1).
  • none of the cysteine residues of the second polypeptide is substituted or deleted.
  • one to three amino acids are deleted at the N- and/or C-terminus of amino acids 296-407 or 299- 407 of human GDF 11 (SEQ ID NO: l).
  • polypeptides described above can be formulated as a pharmaceutical
  • the pharmaceutical composition is a sterile formulation that has a pH of about 5.0 to about 5.5. In certain instances, the pharmaceutical composition is a sterile formulation that has a pH of about 5.5. Such pharmaceutical compositions remain soluble and active when the pH of the formulation is raised to neutral pH (e.g., upon administration to a human subject).
  • the linker is a peptide linker.
  • any arbitrary single-chain peptide comprising about one to 30 residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids) can be used as a linker.
  • the linker is 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 144, or 10 to 150 amino acids in length.
  • the linker contains only glycine and/or serine residues.
  • peptide linkers include: Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO:34); Ser Gly Gly Gly (SEQ ID NO:35); Gly Gly Gly Gly Ser (SEQ ID NO:4); Ser Gly Gly Gly Gly (SEQ ID NO:36); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO:37); Ser Gly Gly Gly Gly Gly (SEQ ID NO:38); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO:39); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO:40); (Gly Gly Gly Gly Ser) n (SEQ ID NO:4)n, wherein n is an integer of one or more; and (Ser Gly Gly Gly Gly)n (SEQ ID NO:36)n, wherein
  • the linker has the amino acid sequence of SEQ ID NO:4 with the exception that the serine residue is replaced with another amino acid. In some instances, the linker has multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the amino acid sequence of SEQ ID NO: 4 with the exception that the serine residue in each copy of the linker is replaced with another amino acid.
  • the linker peptides are modified such that the amino acid sequence GSG (that occurs at the junction of traditional Gly/Ser linker peptide repeats) is not present.
  • the peptide linker comprise an amino acid sequence selected from the group consisting of: (GGGXX)nGGGGS (SEQ ID NO:41) and GGGGS(XGGGS) n (SEQ ID NO:42), where X is any amino acid that can be inserted into the sequence and not result in a polypeptide comprising the sequence GSG, and n is 0 to 4.
  • sequence of a linker peptide is (GGGXiX 2 )nGGGGS and Xi is P and X 2 is S and n is 0 to 4 (SEQ ID NO:43).
  • sequence of a linker peptide is (GGGXiX2)nGGGGS and Xi is G and X2 is Q and n is 0 to 4 (SEQ ID NO:44).
  • sequence of a linker peptide is (GGGXiX2)nGGGGS and Xi is G and X2 is A and n is 0 to 4 (SEQ ID NO:45).
  • sequence of a linker peptide is
  • a linker peptide of the invention comprises or consists of the amino acid sequence
  • a linker peptide comprises or consists of the amino acid sequence (GGGGQ)2GGGGS (SEQ ID NO:48). In yet another embodiment, a linker peptide comprises or consists of the amino acid sequence (GGGPS) 2 GGGGS (SEQ ID NO:49). In a further embodiment, a linker peptide comprises or consists of the amino acid sequence GGGGS(PGGGS) 2 (SEQ ID NO:50).
  • the linker is an XTEN.
  • the linker can be AE
  • the linker is a synthetic compound linker (chemical cross- linking agent).
  • cross-linking agents that are available on the market include N- hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol
  • disulfosuccinimidyl tartrate sulfo-DST
  • bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone BSOCOES
  • bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone sulfo-BSOCOES
  • one or more polypeptides described herein can comprises at least one heterologous moiety that is a "half-life extending moiety.”
  • Half-life extending moieties can comprise, for example, (i) XTEN polypeptides; (ii) Fc; (iii) human serum albumin (HSA), (iv) albumin binding polypeptide or fatty acid, (v) the C-terminal peptide (CTP) of the ⁇ subunit of human chorionic gonadotropin, (vi) proline-alanine-serine polymer (PAS); (vii) homo-amino acid polymer (HAP); (viii) human transferrin; (ix) polyethylene glycol (PEG); (x) hydroxyethyl starch (HES), (xi) polysialic acids (PSAs); (xii) a clearance receptor or fragment thereof which blocks binding of the chimeric molecule to a clearance receptor; (xiii) low complexity peptides
  • XTEN polypeptide Non-limiting, examples of XTENs are disclosed in U.S. Patent
  • the XTEN is AE144. In another embodiment, the XTEN is AE288. In yet another embodiment, the XTEN is AE864.
  • the half-life extending moiety comprises an Fc region.
  • the Fc region comprises the hinge, CH2 and CH3 domains.
  • the Fc region is from IgGl.
  • the Fc region is from IgG2.
  • the Fc region is from IgG4.
  • the Fc region comprises the hinge and CH2 regions from IgG4 and the CH3 domain from IgGl .
  • the hinge region from IgG4 can comprise the S228P mutation.
  • the Fc region may also include one or more substitutions that reduce the effector function.
  • the N-linked glycosylation site of the Fc region is mutated (e.g., T299A, T299C, N297Q).
  • the Fc region may comprise one or both of these mutations: V234A and G237A, that can reduce effector function.
  • the half-life extending moiety comprises two Fc regions fused by a linker.
  • Exemplary heterologous moieties also include, e.g., FcRn binding moieties (e.g., complete Fc regions or portions thereof which bind to FcRn), single chain Fc regions (scFc regions, e.g., as described in U.S. Publ. No.
  • a heterologous moiety can include an attachment site for a non-polypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations of these moieties.
  • PEG polyethylene glycol
  • HES hydroxyethyl starch
  • polysialic acid or any derivatives, variants, or combinations of these moieties.
  • the half-life extending moiety comprises human serum albumin (HSA) or a functional fragment thereof.
  • HSA human serum albumin
  • Examples of albumin or the fragments or variants thereof are disclosed in US Pat. Publ. Nos. US2008/0194481, US2008/0004206, US2008/0161243, US2008/0261877, or US2008/0153751 or PCT Appl. Publ. Nos.
  • the half-life extending moiety can comprise an albumin binding moiety, which comprises an albumin binding peptide, a bacterial albumin binding domain, an albumin-binding antibody fragment, or any combinations thereof.
  • the albumin binding protein can be a bacterial albumin binding protein, an antibody or an antibody fragment including domain antibodies (see, e.g., U.S. Pat. No. 6,696,245).
  • An albumin binding protein for example, can be a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig and Skerra (1998) J. Immunol. Methods 218, 73-83).
  • Other examples of albumin binding peptides that can be used as conjugation partner are, for instance, those described in U.S.
  • the half-life extending moiety can comprise one ⁇ subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin or fragment, variant, or derivative thereof.
  • the half-life extending moiety can comprise a PAS sequence.
  • a PAS sequence as used herein, means an amino acid sequence comprising mainly alanine and serine residues or comprising mainly alanine, serine, and proline residues, the amino acid sequence forming random coil conformation under physiological conditions. Accordingly, the PAS sequence is a building block, an amino acid polymer, or a sequence cassette comprising, consisting essentially of, or consisting of alanine, serine, and proline which can be used as a part of the polypeptides described herein.
  • Non-limiting examples of PAS sequences are disclosed in US Pat. Publ. No. 2010/0292130 and PCT Appl. Publ. No.
  • the half-life extending moiety is a soluble polymer including, but not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, or polyvinyl alcohol.
  • PEG polyethylene glycol
  • the half-life extending moiety is PEG.
  • the polyethylene glycol can have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,
  • the polyethylene glycol can have a branched structure. Branched polyethylene glycols are described, for example, in U. S. Pat. No. 5,643,575; Morpurgo et al, Appl.
  • one or more of the polypeptides described herein can be stabilized by peptide stapling (see, e.g., Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated by reference herein in its entirety). In certain embodiments, one or more of the polypeptides described herein can be stabilized by hydrocarbon stapling.
  • the stapled peptide (e.g., hydrocarbon stapled) is a polypeptide comprising or consisting of the prodomain fragment of GDFl 1 (e.g., 60-112, 60-1 14, or 60-117 of SEQ ID NO: l or comprising 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8) amino acid substitutions, deletions and/or insertions therein).
  • the stapled peptide can include at least two (e.g., 2, 3, 4, 5, 6, 7, 8) amino acid substitutions, wherein the substituted amino acids are separated by two, three, or six amino acids, and wherein the substituted amino acids are non-natural amino acids with olefinic side chains.
  • Peptide stapling is a term coined from a synthetic methodology wherein two olefin- containing side-chains (e.g., cross-linkable side chains) present in a polypeptide chain are covalently joined (e.g., "stapled together") using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, e.g., Blackwell et al., J. Org. Chem., 66: 5291-5302, 2001 ; Angew et al., Chem. Int. Ed. 37:3281, 1994).
  • RCM ring-closing metathesis
  • peptide stapling includes the joining of two (e.g., at least one pair of) double bond-containing side-chains, triple bond-containing side-chains, or double bond-containing and triple bond-containing side chain, which may be present in a polypeptide chain, using any number of reaction conditions and/or catalysts to facilitate such a reaction, to provide a singly “stapled” polypeptide.
  • multiply stapled polypeptides refers to those polypeptides containing more than one individual staple, and may contain two, three, or more independent staples of various spacings.
  • peptide stitching refers to multiple and tandem “stapling” events in a single polypeptide chain to provide a "stitched" (e.g., tandem or multiply stapled) polypeptide, in which two staples, for example, are linked to a common residue.
  • Peptide stitching is disclosed, e.g., in WO 2008/121767 and WO 2010/068684, which are both hereby incorporated by reference in their entirety.
  • staples as used herein, can retain the unsaturated bond or can be reduced. While many peptide staples have all hydrocarbon cross-links, other type of cross-links or staples can be used.
  • triazole-containing (e.g., 1, 4 triazole or 1 , 5 triazole) crosslinks can be used (see, e.g., Kawamoto et al. 2012 Journal of Medicinal Chemistry 55: 1137; WO 2010/060112).
  • Stapling of a peptide using an all-hydrocarbon cross-link has been shown to help maintain its native conformation and/or secondary structure, particularly under physiologically relevant conditions (see, e.g., Schafffle et al, J. Am. Chem. Soc, 122:5891-5892, 2000; Walensky et al, Science, 305 : 1466-1470, 2004).
  • Stapling the polypeptide(s) described herein by an all-hydrocarbon crosslink can improve stability and various pharmacokinetic properties.
  • the stapled polypeptide comprise at least two modified amino acids joined by an internal intramolecular cross-link (or "staple"), wherein the at least two amino acids are separated by 2, 3, or 6 amino acids.
  • Stabilized peptides herein include stapled peptides, including peptides having two staples and/or stitched peptides.
  • the at least two modified amino acids can be unnatural alpha-amino acids (including, but not limited to ⁇ , ⁇ - disubstituted and N-alkylated amino acids). There are many known unnatural amino acids any of which may be included in the peptides of the present invention.
  • unnatural amino acids are 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta- cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N- methyl-L-threonine, N-methyl-L- leucine, 1-amino-cyclopropanecarboxylic acid, 1- amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4- amino-cyclopentenecarboxylic acid, 3-amino- cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-l-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3- diaminopropionic acid, 2,4-diaminobutyric acid, 2- aminoheptanedioic acid, 4- (aminomethyl)benzoic acid, 4-aminomethyl
  • the disclosure features internally cross-linked ("stapled") peptides comprising the amino acid sequence RELRLESIKSQILSKLRLKG (SEQ ID NO:51), wherein the side chains of two amino acids separated by two, three, or six amino acids are replaced by an internal staple; the side chains of three amino acids are replaced by an internal stitch; the side chains of four amino acids are replaced by two internal staples, or the side chains of five amino acids are replaced by the combination of an internal staple and an internal stitch.
  • the stapled peptide can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
  • the stapled polypeptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs:28 to 32, wherein the side chains of two amino acids separated by two, three, or six amino acids are replaced by an internal staple; the side chains of three amino acids are replaced by an internal stitch; the side chains of four amino acids are replaced by two internal staples, or the side chains of five amino acids are replaced by the combination of an internal staple and an internal stitch.
  • the stapled polypeptide comprises or consists of
  • SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL SEQ ID NO:21
  • stapled peptides are: SPRELRXESIXSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL (SEQ ID NO:21)
  • X's in the above sequences can be the same or different non-natural amino acids which can be covalently joined (“stapled together") using a ring-closing metathesis (RCM) reaction to form a cross-linked ring.
  • RCM ring-closing metathesis
  • nucleic acids encoding the polypeptides described herein.
  • amino acid sequences provided herein, one of ordinary skill in the art could easily identify and synthesize nucleic acid sequences encoding these polypeptides.
  • the nucleic acid sequences may include a heterologous sequence or sequences (e.g., a regulatory element such as a promoter, enhancer, ribosome binding site, transcription terminator; a nucleic acid encoding a signal peptide).
  • nucleic acids encoding the polypeptide(s) can be introduced into a host cell by a variety of known methods, e.g., transformation, transfection, electroporation, bombardment with nucleic acid-coated microprojectiles, etc.
  • the nucleic acids encoding the polypeptide(s) can be inserted into a vector appropriate for expression in the host cells before being introduced into the host cells.
  • vectors can contain sequence elements that enable expression of the inserted nucleic acids at the RNA and protein levels.
  • Such expression vectors are well known in the art, and many are commercially available.
  • the vector is a plasmid or a viral vector.
  • the vectors can be introduced into host cells (e.g., microbial cells, yeast cells, insect cells, mammalian cells).
  • host cells e.g., microbial cells, yeast cells, insect cells, mammalian cells.
  • host cells e.g., bacterial cells such as Escherichia coli or Bacillus stear other mophilus, fungal cells such as Saccharomyces cerevisiae or Pichia pastoris, insect cells such as lepidopteran insect cells including Spodoptera frugiperda cells, or mammalian cells such as Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, monkey kidney cells, HeLa cells, human hepatocellular carcinoma cells, or 293 cells, among many others.
  • CHO Chinese hamster ovary
  • BHK baby hamster kidney
  • monkey kidney cells HeLa cells
  • human hepatocellular carcinoma cells or 293 cells
  • the host cell is a fungal cell.
  • the fungal cell is a filamentous fungal cell (e.g., Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei).
  • the host cell is an algal cell, such as a microalgal cell (e.g., Chlamydomonas reinhardtii, Scenedesmus obliquus).
  • the host cells containing the nucleic acids can be cultured under conditions so as to enable the cells to express the nucleic acids.
  • the isolated polypeptide(s) can be formulated as a sterile composition for administration to a human subject.
  • the formulation can have a pH of about 5.0 to about 6.5. In certain instances, the formulation has a pH of about 5.5.
  • the multimeric protein or polypeptide(s) described herein can be used to treat a neurodegenerative disease in a human subject in need thereof.
  • the human subject has a disease or disorder of the central nervous system.
  • the human subject has a disease or disorder of the peripheral nervous system.
  • the method comprises administering to the human subject a therapeutically effective amount of the protein or the pharmaceutical composition described herein.
  • the neurodegenerative disease is Alzheimer's disease.
  • the neurodegenerative disease is Parkinson's disease.
  • the neurodegenerative disease is
  • the neurodegenerative disease is spinal muscular atrophy (SMA), myasthenia gravis, Isaacs syndrome, Stiff-Person syndrome, Guillian-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, amyotrophic lateral sclerosis, peripheral neuropathy, or thoracic outlet compression syndrome.
  • the method involves administering a second agent that is also useful to treat the neurodegenerative disease.
  • the second agent is an antibody or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment is anti-Abeta antibody or antigen-binding fragment (e.g., aducanumab, see, e.g., WO 2008/081008, incorporated by reference herein its entirety).
  • the antibody or antigen-binding fragment is anti-a-synuclein antibody or antigen-binding fragment (see, e.g., WO
  • the antibody or antigen-binding fragment is anti-tau antibody or antigen- binding fragment (see, e.g., WO 2014/100600 and WO 2012/049570, incorporated by reference herein their entirety).
  • the antibody or antigen-binding fragment is anti-TDP-43 antibody antigen-binding fragment (see, e.g., WO 2013/061163, incorporated by reference herein its entirety).
  • the antibody is an anti- LINGO-1 antibody (see, e.g., WO 2008/086006, incorporated by reference herein its entirety).
  • the antibody is an anti-TWEAK antibody (see, e.g., WO 2006/130374, incorporated by reference herein its entirety).
  • the GDF11 polypeptide(s) is conjugated with or administered with a moiety or an agent that allows it traverse the blood brain barrier (e.g., FC5single domain antibody, FC5-Fc, anti-transferrin antibody (e.g., 0X26), insulin-like growth factor-1 receptor antibody, insulin receptor antibody).
  • the multimeric protein or polypeptide(s) described herein can be used to treat an age-related cardiac hypertrophy in a human subject in need thereof.
  • the method comprises administering to the human subject a therapeutically effective amount of the protein or the pharmaceutical composition described herein.
  • the subject has or has been diagnosed with a condition selected from the group consisting of diastolic heart failure, cardiac hypertrophy, an age-related cardiac hypertrophy, hypertension, valvular disease, aortic stenosis, genetic hypertrophic cardiomyopathy, or stiffness of the heart due to aging.
  • the multimeric protein or polypeptide(s) described herein can also be used to treat a muscular or neuromuscular disease or disorder in a human subject in need thereof.
  • the method comprises administering to the human subject a therapeutically effective amount of the protein or the pharmaceutical composition described herein.
  • the muscular or neuromuscular disease or disorder is muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, or cachexia.
  • the multimeric protein or polypeptide(s) described herein can be used to treat a metabolic disease or disorder resulting from abnormal glucose homeostasis.
  • the method comprises administering to the human subject a therapeutically effective amount of the protein or the pharmaceutical composition described herein.
  • the metabolic disease or disorder resulting from abnormal glucose homeostasis is type 2 diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, or obesity.
  • the multimeric protein or polypeptide(s) described herein can be used to treat a human subject suffering from a bone degenerative disorder.
  • the method comprises administering to the human subject a therapeutically effective amount of the protein or the pharmaceutical composition described herein.
  • the bone degenerative disorder is osteoporosis.
  • the multimeric protein or polypeptide(s) described herein can be used to diminish signs of aging.
  • the protein(s) described herein can be used to diminish dermatological signs of aging.
  • the method comprises administering to the human subject (e.g., by topical application to the skin) a therapeutically effective amount of the protein or the pharmaceutical composition described herein.
  • the multimeric protein or polypeptide(s) described herein can be used to treat thymic insufficiency.
  • the protein(s) described herein can be used to induce growth of thymic tissues or thymic epithelial cells.
  • the method comprises administering to the human subject a therapeutically effective amount of the protein or the pharmaceutical composition described herein.
  • the multimeric protein or polypeptide(s) described herein can be administered by any suitable method, e.g., intravenously, subcutaneously, intraperitoneally, intra-arterially, or intra-coronary arterially.
  • GDFl 1 Expression plasmid pACE378 with CMV promoter and encoding the full length gene for human GDFl 1 followed by Ires-mDHFR for selection was engineered for expression in CHO cells. Suspension-adapted CHO cells were transfected with the plasmid and selected for integration by absence of nucleosides from serum-free media. Once established, this stable pool was cryopreserved. For production runs, cultures were expanded in serum-free media up to final volume of 20 L, grown for 4 days to high density (5 x 10 6 cells/ mL) with appropriate feeds, and shifted to a reduced temperature.
  • the column was washed with 20 mM Na2HP04 pH 7.0, 0.5 M NaCl, then with 5 mM NaH 2 P0 4 pH 6.0, 100 mM NaCl, and step eluted with 30 mM, 300 mM, and 500 mM imidazole in 5 mM NaH2P04 pH 6.0, 100 mM NaCl.
  • Column fractions were analyzed for purity by SDS-PAGE. Fractions 26-42 from the 300 mM imidazole elution step (680 mL at 2.7 mg/mL, -1840 mg total protein) were pooled.
  • NaCl and (NH4)2S04 were added to the Ni elution pool to final concentrations of 0.25 M NaCl and 1.2 M (NH4)2S04.
  • the preparation was subjected to centrifugation at 12,000 rpm for 20 min and the clarified supernatant was loaded onto a 120 mL Butyl Sepharose (GE Healthcare) column at room temperature. The column was washed with 5 mM NaH 2 P0 4 pH 6.0, 0.25 M NaCl, 1.2 M (NH 4 ) 2 S0 4 and step eluted with 0.45 M and 0 M (NH 4 ) 2 S0 4 in 5mM NaH 2 P0 4 pH 6.0, 0.25 M NaCl. Column fractions were analyzed for purity by SDS-PAGE.
  • Fractions 4-7 from the 0.45 M (NH 4 ) 2 S0 4 elution step 120 mL, 4.2 mg/mL, 500 mg were pooled.
  • the concentration of GDF1 1 was determined using an extinction coefficient of 1.27 for 1 mg/mL.
  • the protein was dialyzed at 4°C against 5 mM NaH 2 P0 4 pH 6.0, 150 mM NaCl with 4 x 3.5L changes of dialysis buffer, then filtered, ali quoted, and stored at -70°C.
  • GDF1 1 Endoproteinase AspN/Furin Digestion of GDF1 1.
  • GDF1 1 (ACE378, 10 mg, 3.6 mg/mL) in 5 mM NaH 2 P04, 150 mM NaCl, 50 mM Tris HC1 pH 7.5 was treated for 2.5 hr at 37°C with 10 ⁇ g of Endoproteinase AspN (Roche Diagnostics, Indianapolis, IN). NaCl was added to 0.5 M and the sample was loaded onto a 2 mL Ni-Sepharose excel column.
  • the column was washed with 20 mM Na 2 HP0 4 pH 7.0, 0.5 M NaCl, then with NaH 2 P0 4 pH 6.0, 100 mM NaCl, and step eluted with 300 mM imidazole in 5 mM NaH 2 P0 4 pH 6.0, 100 mM NaCl. Elution fractions were pooled and concentrated to ⁇ 3 mL at 1.7 mg/mL. The protein was dialyzed at 4°C against 5 mM NaH 2 P0 4 pH 6.0, 150 mM NaCl with 2 x 1.8L changes of dialysis buffer, aliquoted, and stored at -70°C.
  • Trypsin was obtained from Roche and human plasmin from Sigma. Digestions with trypsin were performed for 5 hr at 1 : 1000 enzyme: GDFl l ratio in 50 mM HEPES pH 8.2, 150 mM NaCl at room temperature and quenched with phenylmethanesulfonyl fluoride (PMSF, Sigma) and leupeptin (Sigma). Digestions with plasmin were performed for 2 hr at 1 :300 enzyme:GDFl 1 ratio in 25 mM HEPES pH 7.5, 150 mM NaCl at room temperature and quenched with PMSF and leupeptin.
  • KIRA Kinase Induced Receptor Activation Assay.
  • the Neuroscreen derivative of PC 12 cells were plated at 2.2 x 10 5 cells/mL per well in 24-well collage Type IV coated plates in Dulbecco's modified eagle medium (DMEM), heat-inactivated 10 % Horse serum, 5 % fetal bovine serum, 4 mM L-glutamine, and cultured overnight for 20 hr at 37°C and 5 % CO2. The medium was dumped out and cells were washed with 1 mL/well phosphate-buffered saline (PBS).
  • DMEM Dulbecco's modified eagle medium
  • PBS phosphate-buffered saline
  • Test samples 300 ⁇ L, were prepared containing 1 : 3 serial dilution of mature GDF 11 (PeproTech, Rocky Hill, NJ) reference standard, full length ACE378 huGDFl l , and AspN- treated ACE378, all starting at 400 ng/mL. 250 of each sample was added to the wells and incubated for 1 hr at 37°C and 5 % CO2. The sample cocktails were dumped out and the cells were washed with 1 mL of PBS.
  • lysis buffer (10 mM Tris HCl, pH 8.0, 0.5 % Nonidet-P40, 0.2 % sodium deoxycholate, 50 mM NaF, 0.1 mM Na 3 V0 4 ,) was added, and after 15 min at room temperature, plates were frozen at -70°C. Samples were analyzed for pSMAD2/3 levels using the PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) sandwich Enzyme-linked Immunosorbent Assay (ELISA) assay kit from Cell Signaling Technology (#12001, Danvers, MA) following the manufacturer's protocol. The required number of microwells for each experiment was broken off after the microwell stripes reached room temperature.
  • PathScan® Phospho-Smad2 Ser465/467
  • Smad3 Smad3
  • ELISA sandwich Enzyme-linked Immunosorbent Assay
  • Luciferase cells were generated by transducing neuroscreen PC 12 cells with lentivirus expressing the firefly luciferase gene under the control of a CMV promoter and SMAD transcriptional response element (TRE) using the Cignal Lenti SMAD reporter (luc) kit CLS- 017L from Qiagen (Germantown, MD). After transduction, the neuroscreen cells were cultured under puromycin selection and once established, the reporter line was cryopreserved.
  • TRE SMAD transcriptional response element
  • the cells were plated at 2.0 x 10 5 cells per well in 24-well collage Type IV coated plates in DMEM, heat- inactivate 10% horse serum, 5% fetal bovine serum, 5 ⁇ g/mL puromycin, and 100 ng/mL NGF, and cultured overnight for 20 hr at 37°C and 5% CO2.
  • the medium was dumped out and cells were washed with 1 mL/well PBS.
  • Serial 1 :3 dilutions of test samples were prepared in 450 DMEM without serum with 100 ng/ml NGF. 250 of each sample was added to the wells and incubated for 5.5 hr at 37°C and 5% CO2.
  • GDF11 (2.9 mg/mL) in 15 mM sodium succinate pH 5.5, 150 mM NaCl was incubated in the dark with 2 mM sodium meta-Periodate (Thermo Scientific) for 30 min at room temperature and immediately desalted on a Zeba spin desalting column equilibrated in 5 mM NaH 2 P04 pH 6.0, 150 mM NaCl (Thermo Scientific).
  • EZ- LinkHydrazide-LC-Biotin (Thermo Scientific) was added to a final concentration of 5 mM from a 50 mM stock prepared in dimethylsulfoxide.
  • HEPES pH 7.2 was added to 50 mM and the sample was incubated in the dark for 2 hr at room temperature and then desalted on a Zeba spin desalting column equilibrated in 5 mM NaH 2 P04 pH 6.0, 150 mM NaCl.
  • 12 mg of biotinylated GDF11 in 5 mM NaH 2 P04, 150 mM NaCl, 50 mM Tris HC1 pH 7.5 was treated for 2.5 hr at 37°C with 6 ⁇ g of Endoproteinase AspN then purified on Ni-Sepharose excel column as described above for the non-biotinylated sample.
  • the protein was dialyzed at 4°C against 5 mM NaH2P04 pH 6.0, 150 mM NaCl with 2, 1 L changes of dialysis buffer, aliquoted, and stored at -70°C.
  • biotinylated GDF11 peptide 60-112/114/-mature GDF11 complex an aliquot of the sample was digested with furin as described above.
  • free biotinylated GDF11 peptide 60-112/114 an aliquot of the biotinylated AspN- treated GDF11 was heated at 95°C for 10 min and centrifuged at top speed for 4 min in an Eppendorf centrifuge.
  • GDF11 peptide 60-112/114 was in the supernatant and the rest of the protein precipitated and was in the pellet fraction.
  • the specificity of labeling of GDF 11 with biotin hydrazide was confirmed by Western blotting using Streptavidin Horseradish peroxidase for detection. Only full length GDF11 and glycopeptide 60-114 were detected and not the major fragments 122-407 or 299-407.
  • the streptavidin tips were presoaked in Octet buffer for 15 min.
  • the tips were loaded into the instrument, washed for 1 min with the buffer, then biotinylated samples and controls were loaded for 5 min.
  • the tips were washed for 1 min and dissociation monitored for 30 min in the presence of 20 ⁇ free biotin.
  • the tips were loaded with biotinylated GDFl 1 and controls, and washed as described above, then the receptor samples were loaded for 15 min and dissociation monitored for 5 min.
  • a model for the N-terminal prodomain peptide-mature GDFl 1 complex was produced as follows: Using the crystal structure of the latent procomplex 3RJR.pdb (Shi et al, Nature, 474:343-349 (201 1)) from the Protein Data Bank (PDB, Berman et al, Nucl. Acids Res. 28:235-242 (2000)), the mature domain of TGF- ⁇ was superposed with that of GDF l 1 (5E4G.pdb, Padyana et al, Acta Crystallogr. F Struct. Biol. Commun. , 72: 160-164 (2016)) using PyMOL (PyMOL, 2012).
  • the GDF1 1 al helix was docked relative to the GDFl 1 mature domain dimer in the active conformation (Padyana et al, ⁇ supra)) using the protein-protein docking software PIPER (Kozakov et al, Biophysical Journal 89(2): 867-875 (2005); Kozakov et al, Proteins: Structure, Function, and
  • N-terminal sequences in the GDFl 1 prodomain contain a putative transmembrane helixlike 13-Ala stretch from Ala-29 to Ala-41.
  • the GDFl l prodomain residues 25-122 may form up to four helices, as compared to two in TGF- ⁇ .
  • a construct encoding full-length human GDFl 1 was expressed in CHO cells and purified from the culture medium by sequential column chromatography steps on Ni Excel and Butyl Sepharose.
  • SDS-PAGE analysis of the purified product revealed a single prominent band with molecular mass of 110 kDa under non-reducing and 55 kDa under reducing conditions consistent with the molecular mass of the GDFl 1 precursor containing both the pro and mature domains and formation of the characteristic homodimer.
  • the 55 kDa band was immunoreactive with anti-GDFl 1 antibody specific for mature GDFl 1 when analyzed by SDS-PAGEAVestern blotting under reducing conditions (Figure 2B).
  • the apparent purity of the full length GDFl 1 was approximately 90%.
  • GDFl 1 was tested for function in a Kinase induced receptor activation (KIRA) assay on PC 12 cells monitoring SMAD 2/3 phosphorylation.
  • Figure 4A shows a time course of GDF 11 -induced SMAD 2/3 phosphorylation. The maximum efficacious response occurred at 60 min.
  • Figure 4B Comparison of the activity of mature GDFl 1 standard versus the recombinant full length protein ( Figure 4B) revealed that the GDFl 1 standard was a potent activator of SMAD 2/3 phosphorylation with an EC50 of 20 ng/mL ( ⁇ 1 nM), but recombinant full length GDFl 1 was inactive, consistent with the need for proteolytic activation (Figure 1).
  • Mature GDFl 1 exists as a disulfide-linked homodimer with apparent molecular weights of 25 kDa and 14 kDa under non-reducing and reducing conditions, respectively.
  • a preparation of mature GDFl 1 standard was included in the analysis (lanes 6 and 7, arrows denote the position of the mature GDFl 1 under reducing and non-reducing conditions).
  • the identity of the fragment was confirmed by mass spectrometry (Figure 5, predicted mass for mature GDFl 1 amino acid residues 299-407 12457.4 Da, observed mass 12458 Da). Following 23 hr of digestion, mature GDFl l was the major cleavage product, present at about 60% of the theoretical yield.
  • the AspN/furin cleavage product was tested for function in PC 12 cells in the SMAD 2/3 phosphorylation assay as well as in a SMAD reporter luciferase assay in which luciferase expression is under control of the SMAD transcriptional response element (TRE) ( Figure 4B and 4C). Both assays confirmed proteolytic activation of the GDFl 1 precursor.
  • the SEC profile showed a broad elution peak with an apparent molecular weight of >50 kDa. No high molecular weight aggregates were detected in the preparation. The >50 kDa size was significantly larger than the expected mass of 25 kDa. In column fractions that contain the peak of the GDF11 (lanes 3-5), mature GDF1 1 (25 kDa non-reducing, 14 kDa reducing) as well as the broad band with molecular weight of 6-16 kDa under reducing and non- reducing conditions were detected. When deglycosylated and analyzed by mass
  • the 6-16 kDa band was identified as a fragment of the prodomain containing amino acids 60-114 and residues 60-112 resulting from secondary cleavage of the same peptide at amino acid 112. ( Figure 5B and C).
  • the identity of the peptides 60-112 and 60- 114 were confirmed by collision-induced dissociation (CID) tandem MS/MS.
  • the 60-114 peptide sequence from GDF1 1 was overlaid onto the published crystal structure of latent TGF- ⁇ (Shi et al., ⁇ supra)), it mapped to a feature coined as the lasso/straight jacket region that wraps around the fingers of the mature domain.
  • the GDF 11 peptide contains the entire al helix, lasso, and a portion of a2 helix that due to truncation may, or may not assume the helical conformation seen in the crystal structure.
  • the al helix is buried in the interface between the growth factor monomers, the lasso forms an extended loop that forms hydrophobic contacts with the tips of the mature domain fingers, and the a2 helix occupies an interface on the surface of finger 2 that overlaps with the type II receptor interface of the BMP members of the TGF- ⁇ superfamily.
  • this interface can be observed in the structure of BMP2 in complex with bone morphogenetic protein receptor type la (BMPRIa) and activin receptor type IIA (ActRIIA), which is deposited under PDB-ID 2GOO.pdb (Allendorph et al,
  • this region of GDFl 1 is likely to bind to the prodomain a2 helix of GDFl 1. Furthermore, the a2 helix is comparatively unchanged between the open conformation of BMP9 (4YCG.pdb, 4YCI.pdb, Mi et al., Proc. Natl. Acad. Sci. U.S.A., 1 12:3710-37152015, Fig.
  • Figure 6A shows a model of the mature GDFl 1/propeptide residue 60-114 complex that takes into account potential structural differences with the latent TGF- ⁇ structure and incorporates the assumption that the GDF l 1 a2 helix occupies the type II receptor binding site.
  • the concave type I receptor binding site provides sufficient space for two helices, as evidenced by the BMP9 prodomain a5 helix, which runs parallel to the wrist helix.
  • the GDF l 1 al helix would have to run antiparallel because it starts at the finger 1 side of the molecule. In none of the docking models that were explored did the al helix settle into the expected parallel position.
  • GDFl 1 peptide 60-112/1 14 is twice as long as the 24-residue minimum inhibitory peptide located within the putative al helix of myostatin (Shi et al, 201 1) that binds with 30 nM affinity and blocks its function (Takayama et al, J. Med. Chem., 58: 1544-1549 (2015)).
  • myostatin and its inhibitory peptide association of GDF1 1 peptide 60- 112/1 14 with mature GDF 11 did not impact its activity and is indistinguishable from the activity of the mature GDF 11 alone ( Figure 4C).
  • Fc fusion proteins of the prodomain designed essentially as described previously (Ge et al, Mo/.
  • GDF 11 The binding characteristics of the GDF 11 peptide 60-1 12/114 for mature GDF11 residues 299-407 and for AspN fragment residues 122-407 were assessed using an Octet Red system. For these studies, full length GDF 11 was biotinylated through the single glycan in full length GDF1 1 (Asn-94), which fortuitously is present in the 60-112/114 peptide (see Figure 1).
  • Fractions corresponding to the complex contained both fluorescently labeled mature GDF11 and labeled 60-112/114 peptide (data not shown). The presence of the peptide in the complex indicates that binding to the receptor does not displace the N-terminal prodomain peptide.
  • plasmin treatment also led to cleavage at a second site within the dibasic KRSRR (SEQ ID NO: 18) recognition sequence to yield a 3 amino acid longer fragment containing residues 296-407 (predicted mass 12856.8 Da, observed mass 12856 Da). With a longer incubation time with plasmin, cleavage at Lys-352 occurred, although it was less reactive than the other sites. Trypsin treatment of the GDFl 1 AspN fragment was less selective (lane 6) than furin or plasmin and lead to cleavage at several additional sites, notably the 30 kDa band and distinct low molecular weight bands that overlapped with the 6- 16 kDa glycopeptide.
  • constructs were designed in two orientations, with the prodomain peptide attached at the N and C terminus of the mature domain.
  • G4S SEQ ID NO:4 spacers of varying lengths were incorporated into the designs between the peptide and mature domain to allow proper assembly of the domains.
  • Some of the constructs contained deletions of up to 11 amino acids from the N-terminus of the peptide that, from the model, did not make contacts with the mature domain.
  • constructs contained additional amino acids at the C-terminus that completed the a2 helix ( Figure 6).
  • FIG. 3 An 8-His (SEQ ID NO:3) tag was engineered at the N- terminus of all the constructs to facilitate their purification.
  • Figure 6B and 6C show different orientations of the model for ACE490, which proved to be one of the more successful designs (discussed below). From the model, the 3XG4S (SEQ ID NO: 19) linker allows for proper contacts between the prodomain and mature domain to form and was predicted to be the minimal size that would allow the domains to assemble.
  • Figure 7A and B show Coomassie stained and western SDS-PAGE profiles from conditioned medium of CHO cells expressing the 11 constructs. Expression of the peptide fusions led to a wide range of measureable titers (Table 2) with levels for two of the constructs exceeding 50 mg/L.
  • a series of 11 prodomain-mature fusion constructs were engineered and expressed in CHO cells. All contained the GDF11 signal peptide linked to an 8 histidine (SEQ ID NO:3) affinity tag with a TEV protease cleavage N- terminal to the designs. G4S (SEQ ID NO:4) spacer sequences of varying lengths were included in the designs. Pro sequences are underlined and mature domain sequences are boldened. Relative titers for each construct were assessed directly from the conditioned medium by Western blotting, using the anti-GDF 11 C-terminal peptide polyclonal antibody for detection (see Figure 7B). As control, a mature domain only construct ACE382 was expressed using the GDF11 signal sequence but no His tag.
  • ACE490 and ACE498 were purified from the condition medium by column chromatography on Ni Excel Sepharose.
  • Non-reducing SDS-PAGE analyses of the purified preparations ( Figure 8B and D) of purified preparations of ACE490 and ACE498 revealed that neither sample had formed the interchain disulfide that covalently links the mature GDF1 ldimer when it is assembled properly. Further assessment by SEC showed that both products eluted from the sizing column as monomers (data not shown). When these samples were tested for function in the reporter assay, they were both inactive (see Figures 8C and 8E). Next it was determined if dimer formation could be promoted by redox.
  • FIG 8 A shows a schematic summarizing the redox steps. This treatment of ACE490 led to significant dimer formation.
  • Figure 8B shows an SDS-PAGE analysis of the redox product. No dimer formation occurred without the aldrithiol activation step.
  • GDF 1 1 propeptide NH2-
  • SPRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL SEQ ID NO:21
  • a 10 mg/mL stock solution of the peptide was prepared in 20 mM sodium phosphate pH 7.0, 150 mM NaCl.
  • Carrier free mature GDFl 1 (R&D systems) was reconstituted at 400 ⁇ g/mL in 4 mM HCl. 2 ⁇ g (5 ⁇ ) of the mature GDF l 1 was diluted with 5 ⁇ .
  • Synthetic peptides were designed to better understand how the propeptides from the AspN digest bind to the GDF1 1 mature domain.
  • candidate peptides should be shorter than the 55 residues observed in the AspN digest, up to a range of 30-45 residues.
  • GDF1 1 prodomain peptide 60-1 14 six peptide variants of different lengths were designed ( Figure 11), which included or excluded the latency lasso and the helix or helices described below.
  • Human GDF11 very likely has more than two helices before the AspN cleavage site.
  • PSIPRED (2013) predicts a third helix located between these two helices. Furthermore, when submitting the complete first 132 residues of the human GDF11 prodomain to the same prediction methods ( Figure 12), both methods predict another a-helix for the 13-Ala putative transmembrane helix segment 29-41 ( Figure 13) of the GDFl l prodomain and the additional helix between the al and a2 helices, thus suggesting a total of four a-helices in the prodomain before the AspN cleavage site.
  • Peptides were designed that included the presumed al- and/or a2-helices of the porcine TGF- ⁇ pro-domain/mature domain complex structure (3RJR.pdb, Shi et al, Nature, 474:343-349 (2011)), and the putative helix in between the former two. Based on a multiple sequence alignment of 33 TGF- ⁇ family members, which include GDF11 and myostatin (GDF8), it was suggested that helices homologous to al and a2 are present throughout the family. In porcine TGF- ⁇ , these helices are connected through an unstructured loop, termed the "latency lasso", whose contacts with the mature domain's fingers 1 and 2 likely contribute to binding.
  • the lasso region is rich in proline (6 out of 15 residues, or 40%), which interact with various tryptophans of the mature domain.
  • the latency lasso of GDF11 is six residues longer than the equivalent region of porcine TGF- ⁇ , indicating structural differences.
  • secondary structure prediction methods and a Blast search predict helical content for the latency lasso-equivalent region of human GDF11 while this is not the case for the latency lasso region of TGF- ⁇ .
  • GDFl l 's lasso region while still proline- rich (4 out of 21, or 19%, vs. a 5% average value for vertebrate proteins), has fewer prolines than porcine TGF- ⁇ . These prolines may function as helix breakers or starters, or for interactions with the mature domain as in TGF- ⁇ . The potential occurrence of this additional "lasso helix" was taken into account in the peptide designs.
  • N-terminal and C-terminal helix caps are reported to stabilize monomeric helix formation by up to 2 kcal/mol, while there is no discemable energy advantage for C-caps (Pace et al., Biophysical J, 75, 422-427 (1998)). Helix capping can follow one of seven commonly observed short-range
  • conformational patterns formed by the local sequence preceding or following the a-helix three N-terminal and four C-terminal helix caps), or the helix can be capped by long-range intra-molecular or intermolecular interactions, see Aurora and Rose (Aurora and Rose, Protein Science, 7, 21-38 (1998)).
  • the first N-terminal pattern classified by Aurora and Rose has been called the "SXXE box", or "the hydrophobic staple” (Pace et al, (supra)), in which the polar S is the N-cap, without a preceding hydrophobic residue.
  • the latter pattern was applied.
  • a C-capping glycine was also employed.
  • peptides have a shortened al helix to prune potentially non-helical residues from the helical segment.
  • Four other peptides extend beyond the al helix in case additional helices are essential for binding, or in case some apparently disordered regions are needed for binding or because they form natural helix capping motives.
  • the peptides are set forth in Figure 11.
EP17832636.9A 2016-12-16 2017-12-15 Stabilisierter proteolytisch aktivierter wachstumsdifferenzierungsfaktor 11 Pending EP3562840A1 (de)

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